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Ko ngā kawe ira mō te rongoā i te mate pūkahukahu cystic fibrosis me aro ki ngā ara rere nā te mea kāore te whakawhiti ira o te pūkahukahu ā-taha e whakarato i te painga rongoā. He hononga tika te whai huatanga o te whakawhiti huaketo ki te wā noho o te kawe. Heoi, ko ngā wai tuku pēnei i ngā kawe ira ka horapa noa ki roto i ngā alveoli i te wā e manawa ana, ā, ka tere te whakakore i ngā matūriki rongoā o tētahi āhua mā te kawe mucociliary. He mea nui te whakaroa i te wā noho o ngā kawe ira i roto i ngā ara rere engari he uaua ki te whakatutuki. Ka taea e ngā matūriki aukume honohono-kawe ira ka taea te tuku ki te mata o ngā ara rere te whakapai ake i te arotahi ā-rohe. Nā ngā wero o te tirohanga in vivo, kāore i te tino mārama te whanonga o aua matūriki aukume iti i runga i te mata ara rere i te aroaro o tētahi papa aukume kua whakamahia. Ko te whāinga o tēnei rangahau ko te whakamahi i te whakaahua synchrotron hei whakaatu i te nekehanga in vivo o tētahi raupapa matūriki aukume i roto i te trachea o ngā kiore kua whakamoea hei tirotiro i ngā hihiri me ngā tauira o te whanonga matūriki takitahi me te rahi i roto i te vivo. I muri mai ka aromatawaihia e mātou mēnā ka whakanui ake te tukunga o ngā matūriki aukume lentiviral i te aroaro o tētahi papa aukume i te whai huatanga whakawhiti i roto i te trachea kiore. Hiko-X Synchrotron E whakaatu ana te whakaahua i te whanonga o ngā matūriki aukume i roto i ngā papa aukume tūmau me ngā papa aukume nekeneke i roto i te vitro me te in vivo. Kāore e taea te tōia ngāwari ngā matūriki i te mata o te ara manawa ora me ngā aukume, engari i te wā e kawe ana, ka kohikohia ngā putunga ki te tirohanga kei reira te papa aukume kaha rawa atu. I piki ake hoki te whai huatanga o te whakawhiti ira e ono ngā wā i te wā i tukuna ai ngā matūriki aukume lentiviral i te aroaro o te papa aukume. I te taha o ēnei hua, e kī ana ko ngā matūriki aukume lentiviral me ngā papa aukume he huarahi whai hua pea hei whakapai ake i te arotahi ira me te whakanui ake i ngā taumata whakawhiti ira i roto i te ara manawa i roto i te vivo.
Ko te mate pukupuku kiriuhi (CF) ka puta mai i te rerekētanga o te ira kotahi e kiia nei ko te kaiwhakahaere kawe whakawhiti-membrane CF (CFTR). Ko te pūmua CFTR he hongere katote kei roto i te maha o ngā pūtau epithelial puta noa i te tinana, tae atu ki ngā ara rere, he wāhi nui o te pathogenesis CF. Ko ngā hapa CFTR ka arahi ki te kawe wai rerekē, ka maroke te mata o te ara rere, ka whakaiti i te hohonutanga o te paparanga wai o te mata ara rere (ASL). Ka whakararu hoki tēnei i te kaha o te pūnaha kawe mucociliary (MCT) ki te whakakore i ngā matūriki me ngā pathogens i roto i ngā ara rere. Ko tā mātou whāinga he whakawhanake i tētahi rongoā ira lentiviral (LV) hei tuku i te tārua tika o te ira CFTR me te whakapai ake i te ASL, MCT, me te hauora o ngā pukahukahu, me te whakawhanake tonu i ngā hangarau hou e taea ai te ine i ēnei tawhā i roto i te tinana ora1.
Ko ngā kawe LV tētahi o ngā kaitono matua mō te rongoā ira CF, nā te mea ka taea e rātou te whakauru pumau i te ira rongoā ki roto i ngā pūtau pūtake o te ara manawa (ngā pūtau take ara manawa). He mea nui tēnei nā te mea ka taea e rātou te whakahoki mai i te makuku me te whakakorenga huware mā te wehewehe ki ngā pūtau mata ara manawa e hono ana ki te CF kua whakatikatikaina e te ira mahi, ka hua ake he painga mō te wa katoa. Me anga atu ngā kawe LV ki te ara manawa, nā te mea koinei te wāhi ka tīmata ai te mate pūkahukahu CF. Ko te tukunga o te kawe ki roto i te pūkahukahu ka hua ake pea he whakawhiti alveolar, engari kāore he painga rongoā i roto i te CF. Heoi, ko ngā wai pēnei i ngā kaikawe ira ka heke noa ki ngā alveoli i te wā e manawahia ana i muri i te whānautanga3,4 ā, ka tere te whakakore i ngā matūriki rongoā ki roto i te kōhao waha e te MCT. Ko te whai huatanga o te whakawhiti LV e pā tika ana ki te roa o te wā e noho ana te kawe ki te taha o ngā pūtau ūnga kia taea ai te tango pūtau - te "wā noho"5 - ka ngāwari te whakaitihia e te rere o te hau ā-rohe me te hopu huware matūriki kua whakaritea me te MCT. Mō te CF, he mea nui te kaha ki te whakaroa i te wā noho o te LV i roto i te ara manawa kia tutuki ai ngā taumata whakawhiti teitei i tēnei rohe, engari he uaua rawa atu i tēnei wā.
Hei whakakore i tēnei arai, e kī ana mātou kia rua ngā huarahi e āwhina tahi ai ngā matūriki aukume LV (MP). Tuatahi, ka taea te ārahi mā te aukume ki te mata o te ara manawa hei whakapai ake i te arotahi me te āwhina i ngā matūriki kawe ira kia noho ki te rohe ara manawa e hiahiatia ana; me te ASL) kia neke ki te paparanga pūtau 6. Kua whakamahia whānuitia ngā MP hei waka tuku rongoā arotahi ina herea ki ngā paturopi, ki ngā rongoā chemotherapy, ki ētahi atu ngota iti rānei e piri ana ki ngā kiriuhi pūtau, e herea rānei ki ngā kaiwhakawhiwhi mata pūtau e tika ana, ā, ka kohia ki ngā wāhi pukupuku i te aroaro o te hiko pateko. Ngā Papa Aukume mō te Maimoatanga Mate Pukupuku 7. Ko ētahi atu tikanga "hyperthermal" e whai ana ki te whakamahana i ngā MP ina pāngia rātou e ngā papa aukume hurihuri, ā, ka whakangaro i ngā pūtau pukupuku. Ko te kaupapa o te whakawhiti aukume, e whakamahia ana he papa aukume hei kaihoko whakawhiti hei whakarei ake i te whakawhiti o te DNA ki ngā pūtau, he mea whakamahi noa i roto i te vitro mā te whakamahi i te whānuitanga o ngā ira kore-huaketo me te huaketo mō ngā raina pūtau uaua ki te whakawhiti. Kua whakapumautia te whai huatanga o te whakawhiti aukume LV, me te tuku i roto i te vitro o ngā LV-MP ki te raina pūtau epithelial bronchial tangata i te aroaro o te papa aukume tūmau, e whakanui ana i te pai o te whakawhiti mā te 186-ngā wā ki te whakataurite ki te whakawhiti LV anake. Kua whakamahia hoki te LV-MP ki tētahi tauira CF in vitro, i reira i whakanui ake ai te whakawhiti aukume i te whakawhiti LV i roto i ngā ahurea atanga hau-wai mā te 20-ngā wā i te aroaro o te huware CF10. Heoi, he iti noa te aro ki te whakawhiti aukume in vivo o ngā whekau, ā, kua arotakehia noa i roto i ētahi rangahau kararehe11,12,13,14,15, inā koa i roto i te ngā pukahukahu16,17. Ahakoa rā, he mārama ngā whai wāhitanga mō te whakawhiti aukume i roto i te rongoā pukahukahu CF. I kī a Tan et al.(2020) "mā te rangahau taunakitanga mō te tuku pūkahukahu nanoparticle aukume whai hua ka whakatakoto i te ara mō ngā rautaki manawa CFTR ā muri ake nei hei whakapai ake i ngā putanga haumanu i roto i ngā tūroro CF"6.
He uaua ki te tiro me te ako i te whanonga o ngā matūriki aukume iti i runga i ngā mata o te ara manawa i te aroaro o tētahi papa aukume kua whakamahia, ā, nō reira kāore i te tino mārama. I roto i ētahi atu rangahau, i whakawhanakehia e mātou he tikanga whakaahua X-ray ā-wāhanga-whakawhitiwhiti-synchrotron (PB-PCXI) hei tiro me te ine i ngā huringa iti i roto i te tinana o te hōhonutanga ASL18 me te whanonga MCT19,20 hei ine tika i te makuku o te mata o te awa hau, ā, hei tohu wawe mō te whai huatanga o te maimoatanga. Hei tāpiri, ko tā mātou tikanga aromatawai MCT e whakamahi ana i ngā matūriki 10–35 µm te whānui i hangaia mai i te alumina, i te karāhe taupū whakaata teitei rānei hei tohu MCT e kitea ana mā te whakamahi i te PB-PCXI21. He pai ngā tikanga e rua mō te tiro i ngā momo matūriki, tae atu ki te MP.
Nā te teitei o te taumira ā-wāhi me te ā-wā, he pai rawa ā mātou tikanga tātari ASL me MCT e hangai ana ki te PB-PCXI mō te tirotiro i ngā hihiri me ngā tauira o te whanonga matūriki takitahi me te rahi i roto i te tinana ora hei āwhina i a mātou ki te mārama me te arotau i ngā tikanga tuku ira MP. Ko te huarahi e whakamahia ana e mātou i konei i ahu mai i ā mātou rangahau e whakamahi ana i te raina hihi SPring-8 BL20B2, i reira i whakaatuhia ai e mātou te nekehanga wai i muri i te tuku horopeta vector rūpahu ki roto i ngā ara manawa o te ihu me te pūkahukahu o ngā kiore hei āwhina i te whakamārama i ā mātou tauira whakaaturanga ira kore-taurite i kitea i roto i ā mātou rangahau kararehe horopeta kaikawe ira 3,4.
Ko te whāinga o tēnei rangahau he whakamahi i te synchrotron PB-PCXI hei whakaatu i ngā nekehanga in vivo o tētahi raupapa MP i roto i te trachea o ngā kiore ora. I hangaia ēnei rangahau whakaahua PB-PCXI hei whakamātautau i te whānuitanga o ngā MP, ngā kaha o te papa aukume, me ngā wāhi hei whakatau i tōna pānga ki te nekehanga MP. I whakapae mātou ka āwhina te papa aukume i tukuna ki waho i te MP i tukuna kia noho, kia neke rānei ki te wāhi ūnga. I taea hoki e ēnei rangahau te tautuhi i ngā whirihoranga aukume e whakanui ana i te maha o ngā matūriki e puritia ana i roto i te trachea i muri i te whakatakotoranga. I roto i tētahi raupapa rangahau tuarua, i whai mātou ki te whakamahi i tēnei whirihoranga tino pai hei whakaatu i te tauira whakawhiti i puta mai i te tuku in vivo o ngā LV-MP ki te ara manawa o te kiore, i runga i te whakaaro ko te tukunga o ngā LV-MP i roto i te horopaki o te arotahi ara manawa ka hua ake he pai ake te whai huatanga o te whakawhiti LV.
I whakahaerehia ngā rangahau kararehe katoa i runga anō i ngā tikanga i whakaaetia e te Whare Wānanga o Adelaide (M-2019-060 me M-2020-022) me te SPring-8 Synchrotron Animal Ethics Committee. I whakahaerehia ngā whakamātautau i runga anō i ngā aratohu a ARRIVE.
I mahia ngā whakaahua hihi-X katoa i te raina hihi BL20XU i te synchrotron SPring-8 i Hapani, mā te whakamahi i tētahi whakaritenga rite ki te mea i whakaahuatia i mua21,22. I roto i te poto, i whakanohoia te pouaka whakamātautau ki te 245 m mai i te mowhiti rokiroki synchrotron. Ka whakamahia he tawhiti tauira-ki-te-pūoko o te 0.6 m mō ngā rangahau whakaahua matūriki me te 0.3 m mō ngā rangahau whakaahua in vivo hei whakaputa i ngā pānga rerekētanga ā-wāhanga. I whakamahia he pūngao hihi kotahi-tae o te 25 keV. I hopukina ngā whakaahua mā te whakamahi i tētahi kaitahuri hihi-X taumira teitei (SPring-8 BM3) i honoa ki tētahi pūoko sCMOS. Ka hurihia e te pūoko ngā hihi-X ki te mārama kitea mā te whakamahi i tētahi scintillator matotoru 10 µm (Gd3Al2Ga3O12), ka tukuna ki tētahi pūoko sCMOS mā te whakamahi i tētahi whāinga karuārai × 10 (NA 0.3). Ko te pūoko sCMOS ko Orca-Flash4.0 (Hamamatsu Photonics, Hapani) me te rahi o te huinga o 2048 × 2048 pika me te rahi pika mata o te 6.5 × 6.5 µm. Mā tēnei tatūnga ka puta he rahi pika isotropic whai hua o te 0.51 µm me te tirohanga whānui o te 1.1 mm × 1.1 mm. I whiriwhiria he roa whakaaturanga o te 100 ms hei whakanui ake i te ōwehenga tohu-ki-haruru o ngā matūriki aukume i roto me waho o te ara manawa, me te whakaiti i ngā mahi nekehanga i puta mai i te manawa. Mō ngā rangahau in vivo, i whakanohoia he kati hihi-X tere ki te ara hihi-X hei whakawhāiti i te horopeta irahiko mā te aukati i te hihi hihi-X i waenga i ngā whakaaturanga.
Kāore i whakamahia te kawe LV i roto i ngā rangahau whakaahua SPring-8 PB-PCXI nā te mea kāore te rūma whakaahua BL20XU i whai tiwhikete Taumata Haumaru Koiora 2. Engari, i tīpakohia e mātou he whānuitanga o ngā MP kua tautuhia pai mai i ngā kaiwhakarato arumoni e rua—e kapi ana i te whānuitanga o ngā rahi, ngā rauemi, ngā kukū rino, me ngā tono—tuatahi kia mārama ai ki te pānga o ngā papa aukume ki te nekehanga MP i roto i ngā uaua karāhe, kātahi ki roto i ngā ara manawa ora. i runga i te mata. Ko te rahi o ngā MP kei waenga i te 0.25 ki te 18 μm, ā, he maha ngā rauemi i hangaia ai (tirohia te Ripanga 1), engari kāore i te mōhiotia te hanganga o ia tauira, tae atu ki te rahi o ngā matūriki aukume i roto i te MP. I runga i ā mātou rangahau whānui MCT 19, 20, 21, 23, 24, e tumanakohia ana ka kitea ngā MP iti iho i te 5 μm i runga i te mata o te ara manawa tracheal, hei tauira mā te tango i ngā anga karapīpiti hei kite i te tirohanga pai ake o te nekehanga MP. He iti ake te MP kotahi te rahi 0.25 μm i te taumira o te taputapu whakaahua, engari e tumanakohia ana ka kitea e te PB-PCXI tō rātou rerekētanga rōrahi me te nekehanga o te wai mata e whakatakotoria ana i muri i te whakatakotoranga.
I whakaritea ngā tauira mō ia MP i te Ripanga 1 i roto i ngā pūwero karāhe 20 μl (Drummond Microcaps, PA, USA) me te diameter o roto o te 0.63 mm. Kei roto i te wai ngā matūriki corpuscular, ko ngā matūriki CombiMag kei roto i te wai motuhake a te kaihanga. Kei te haurua o ia ngongo te whakakī ki te wai (tata ki te 11 μl) ka whakatakotoria ki runga i te kaipupuri tauira (tirohia te Pikitia 1). I whakatakotoria ngā pūwero karāhe whakapae ki runga i te atamira tauira i roto i te pouaka whakaahua, ia, ā, i whakanohoia ngā taha o te wai. I tāpirihia he aukume anga nikara 19 mm te diameter (28 mm te roa) neodymium rino boron (NdFeB) (N35, nama kāt. LM1652, Jaycar Electronics, Ahitereiria) me te aukume toenga o te 1.17 Tesla ki tētahi atamira whakawhiti motuhake hei whakatutuki i te Huri i tōna tūranga mamao i te wā whakaahua. Ka tīmata te hopu whakaahua X-ray ina whakanohoia te aukume ki te 30 mm i runga ake i te tauira, ā, ka riro mai ngā whakaahua i te tere o te 4 anga ia hekona. I te wā whakaahua, i whakatatahia te aukume ki te ngongo pūwero karāhe. (tata ki te 1 mm te tawhiti) kātahi ka whakamāoritia i te taha o te ngongo hei aromatawai i ngā pānga o te kaha me te tūnga o te mara.
Tatūnga whakaahua in vitro kei roto ngā tauira MP i roto i ngā uaua karāhe i te atamira whakamāoritanga xy. Kua tohua te ara o te hihi X-ray ki te rārangi whero pītiti.
I te wā i whakatūria ai te tirohanga in vitro o ngā MP, i whakamatautauria tētahi wāhanga iti o rātou in vivo i roto i ngā kiore Wistar wahine albino momo mohoao (~12 wiki te pakeke, ~200 g). 0.24 mg/kg medetomidine (Domitor®, Zenoaq, Hapani), 3.2 mg/kg midazolam (Dormicum®, Astellas Pharma, Hapani) me te 4 mg/kg butorphanol (Vetorphale®, Meiji Seika) I whakamoea ngā kiore ki te ranunga o Pharma), Hapani) mā te werohanga intraperitoneal. Whai muri i te whakamoea, i whakaritea rātou mō te whakaahua mā te tango i te huruhuru huri noa i te trachea, te whakauru i tētahi ngongo endotracheal (ET; 16 Ga iv cannula, Terumo BCT) me te pupuri i a rātou kia takoto ki runga i tētahi pereti whakaahua ritenga kei roto he putea wera hei pupuri i te pāmahana o te tinana 22. Kātahi ka tāpirihia te pereti whakaahua ki te wāhanga whakamaoritanga tauira i roto i te pouaka whakaahua i tētahi koki paku hei whakarārangi i te trachea whakapae i roto i te whakaahua hihi-X, e whakaaturia ana i roto i te Pikitia 2a.
(a) I te whakatakotoranga whakaahua in vivo i roto i te pouaka whakaahua SPring-8, kua tohua te ara o te hihi-X ki te rārangi whero kua pakaru. (b,c) I mahia te whakanoho aukume ki runga i te trachea mai i tawhiti mā te whakamahi i ngā kāmera IP e rua kua whakanohoia ki te taha orthogonal. I te taha maui o te whakaahua mata, ka kitea te koropiko waea e pupuri ana i te pane, me te cannula tuku kei roto i te ngongo ET.
I honoa he pūnaha papu werowero mamao (UMP2, World Precision Instruments, Sarasota, FL) mā te whakamahi i te werowero karāhe 100 μl ki te ngongo PE10 (OD 0.61 mm, ID 0.28 mm) mā te ngira 30 Ga. Tohua te ngongo kia tika ai te tūranga o te pito i roto i te trachea i te wā e whakaurua ana te ngongo ET. Mā te whakamahi i te papu iti, i tangohia te pūwero werowero i te wā e rumakina ana te pito o te ngongo ki roto i te tauira MP hei tuku. Kātahi ka whakaurua te ngongo tuku kua utaina ki roto i te ngongo endotracheal, ka whakanoho i te pito ki roto i te wāhanga kaha rawa atu o tā mātou papa aukume e tumanakohia ana. I whakahaeretia te hopu whakaahua mā te whakamahi i te taputapu kite manawa e hono ana ki tā mātou pouaka wā Arduino, ā, i tuhia ngā tohu katoa (hei tauira, te pāmahana, te manawa, te whakatuwheratanga/kati o te kati me te hopu whakaahua) mā te whakamahi i te Powerlab me te LabChart (AD Instruments, Sydney, Ahitereiria) 22. I te wā e whakaahua ana I te wā kāore e taea te uru atu ki te whare, e rua ngā kāmera IP (Panasonic BB-SC382) i whakanohoia ki te 90° ki a rātou anō, ā, i whakamahia hei aroturuki i te tūranga. o te aukume e pā ana ki te trachea i te wā whakaahua (Pikitia 2b,c). Hei whakaiti i ngā mahi nekehanga, kotahi te whakaahua i tangohia mō ia manawa i te wā o te papatairite rere o te tai.
Ka tāpirihia he aukume ki tētahi wāhanga tuarua ka taea te whakanoho mai i waho o te whare whakaahua. I whakamātauria ngā tūranga me ngā whirihoranga aukume maha, tae atu ki: I whakairihia ki tētahi koki tata ki te 30° i runga ake i te trachea (ngā whirihoranga e whakaaturia ana i roto i ngā Pikitia 2a me te 3a); tētahi aukume i runga ake i te kararehe, ko tētahi i raro iho, me ngā pou kua whakaritea hei kukume (Pikitia 3b); tētahi aukume i runga ake i te kararehe, ko tētahi i raro iho, me ngā pou kua whakaritea hei pana (Pikitia 3c); me tētahi aukume i runga ake, ā, he poutū ki te trachea (Pikitia 3d). Kia oti te whirihora i te kararehe me te aukume, ā, kia utaina te MP hei whakamātautau ki roto i te papu weronga, tukuna he horopeta 50 μl i te tere o te 4 μl/hēkona i a koe e kohi whakaahua ana. Kātahi ka nekehia te aukume ki mua, ki muri rānei i te trachea, i a koe e kohi whakaahua ana.
Te whirihoranga aukume mō te whakaahua in vivo (a) he aukume kotahi i runga ake i te trachea i te koki o te 30° pea, (b) e rua ngā aukume kua whakaritea hei kukume, (c) e rua ngā aukume kua whakaritea hei pana, (d) he aukume kotahi i runga ake, ā, he poutū i roto i te trachea. I titiro te kaimātakitaki mai i te waha ki ngā pukahukahu mā roto i te trachea, ā, ka puta te hihi X-ray mā te taha maui o te kiore, ka puta atu i te taha matau. Ka nekehia te aukume i te roanga o te ara manawa, i te taha maui, matau rānei i runga ake i te trachea i te ahunga o te hihi X-ray.
I whai hoki mātou ki te whakatau i te tirohanga me te whanonga o ngā matūriki i roto i ngā ara manawa i te korenga o te manawa whakararuraru me te nekehanga o te ngākau. Nō reira, i te mutunga o te wā whakaahua, i patua ngā kararehe i runga i te atawhai mō te nui rawa o te pentobarbital (Somnopentil, Pitman-Moore, Washington Crossing, USA; ~65 mg/kg ip). I waiho ētahi kararehe ki runga i te papa whakaahua, ā, i te mutunga o te manawa me te ngakau o te ngākau, ka tuaruatia te tukanga whakaahua, me te tāpiri i tētahi atu horopeta o te MP mēnā kāore he MP e kitea ana i runga i te mata o te ara manawa.
I whakatikatikaina ngā whakaahua i kohia ki te papatahi me te papapōuri, kātahi ka huihuia hei kiriata (20 anga ia hekona; 15-25 × tere noa i runga i te tere manawa) mā te whakamahi i tētahi tuhinga ritenga i tuhia ki MATLAB (R2020a, The Mathworks).
I whakahaerehia ngā rangahau tuku ira LV katoa i te Whare Rangahau Kararehe Taiwhanga i Te Whare Wānanga o Adelaide, ā, ko te whāinga he whakamahi i ngā hua o te whakamātautau SPring-8 hei aromatawai mēnā ka taea e te tuku LV-MP i te aroaro o te papa aukume te whakarei ake i te whakawhiti ira in vivo. Hei aromatawai i ngā pānga o te MP me te papa aukume, e rua ngā rōpū kararehe i rongoatia: i hoatu ki tētahi rōpū he LV-MP me te aukume kua whakanohoia, ā, i whiwhi tētahi atu rōpū i tētahi rōpū whakahaere me te LV-MP kāore he aukume.
I hangaia ngā ira kawe LV mā te whakamahi i ngā tikanga kua whakamāramahia i mua 25, 26. Ko te ira kawe LacZ e whakaatu ana i te ira beta-galactosidase kua whakatōkia ki te karihi e whakahaerehia ana e te kaiwhakatairanga MPSV (LV-LacZ), e whakaputa ana i te hua tauhohenga kikorangi i roto i ngā pūtau kua whakawhitia, e kitea ana i roto i ngā kiko o mua o te pukahukahu me ngā wāhanga kiko. I mahia te titration i roto i ngā ahurea pūtau mā te tatau ā-ringa i te maha o ngā pūtau LacZ pai me te hemocytometer hei tatau i te titer i roto i te TU/ml. Ka tiakina ngā kaikawe ki te -80 °C, ka rewa i mua i te whakamahinga, ā, ka herea ki te CombiMag mā te whakaranu i te ōwehenga 1:1 me te whakatō ki runga i te hukapapa mō te 30 meneti pea i mua i te tukunga.
I whakamoea ngā kiore Sprague Dawley noa (n = 3/rōpū, ~2-3 i roto i te puku ki te ranunga o te 0.4 mg/kg medetomidine (Domitor, Ilium, Ahitereiria) me te 60 mg/kg ketamine (Ilium, Ahitereiria) marama-te-pakeke) ip) werohanga me te cannulation ā-waha kore-pokanga me te cannula 16 Ga iv. Hei whakarite kia whiwhi te kiko ara manawa tracheal i te transduction LV, i whakatikatikaina mā te whakamahi i tā mātou tikanga whakararuraru miihini kua whakaahuahia i mua, i roto i tēnei i mirimirihia te mata ara manawa tracheal ki te taha tuaka ki te kete waea (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28. Kātahi ka whakahaerehia te whakahaerenga o te LV-MP ki roto i te tracheal i roto i te kāpata haumaru koiora tata ki te 10 meneti i muri i te whakararuraru.
I whirihorahia te papa aukume i whakamahia i roto i tēnei whakamātautau i roto i te huarahi rite ki te rangahau whakaahua X-ray in vivo, me ngā aukume ōrite i puritia i runga ake i te trachea mā te whakamahi i ngā topenga stent whakahoro (Pikitia 4). I tukuna he rōrahi 50 μl (2 × 25 μl aliquots) o LV-MP ki roto i te trachea (n = 3 kararehe) mā te whakamahi i tētahi pipette kei roto he pito gel e ai ki te whakaahuatanga o mua. I whiwhi tētahi rōpū whakahaere (n = 3 kararehe) i ngā LV-MP ōrite me te kore e whakamahi i tētahi aukume. I muri i te otinga o te werohanga, ka tangohia te cannula mai i te ngongo ET ka tangohia te kararehe. Ka noho te aukume ki tōna wāhi mō te 10 meneti, kātahi ka tangohia. I whiwhi ngā kiore i tētahi horopeta meloxicam (1 ml/kg) i raro i te kiri (Ilium, Ahitereiria) ā, muri iho ka hurihia te rongoā whakamoe mā te werohanga ip o te 1 mg/kg atipamazole hydrochloride (Antisedan, Zoetis, Ahitereiria). I puritia ngā kiore kia mahana, ā, i aroturukitia kia ora katoa mai i te rongoā whakamoe.
He taputapu tuku LV-MP i roto i tētahi kāpata haumaru koiora. Ka kitea te pū Luer hina mārama o te ngongo ET e puta mai ana i te waha, ā, ko te pito tiēre o te pipette e whakaaturia ana i te pikitia ka whakaurua mā te ngongo ET ki te hohonutanga e hiahiatia ana ki roto i te trachea.
Kotahi wiki i muri i te tikanga inenga LV-MP, i patua ngā kararehe mā te manawa CO2 100%, ā, i aromatawaihia te whakaaturanga LacZ mā te whakamahi i tā mātou maimoatanga X-gal paerewa. I tangohia ngā mowhiti e toru o te hope, te nuinga o te wheua, kia kore ai e whakaurua ki te tātari tetahi kino ā-tinana, tetahi pupuri wai rānei mai i te whakatakotoranga ngongo endotracheal. I tapahia ia trachea kia rua ngā haurua hei hanga i ngā haurua e rua mō te tātari, ā, i whakanohoia ki roto i tētahi rihi kei roto te rapa silicone (Sylgard, Dow Inc) mā te whakamahi i te ngira Minutien (Fine Science Tools) hei tiro i te mata luminal. I whakaūhia te tohatoha me te tauira o ngā pūtau i whakawhitihia mā te whakaahua o mua mā te whakamahi i te karuārai Nikon (SMZ1500) me te kāmera DigiLite me te pūmanawa TCapture (Tucsen Photonics, Haina). I tangohia ngā whakaahua i te whakanui 20x (tae atu ki te tautuhinga teitei rawa atu mō te whānui katoa o te trachea), me te whakaahua i te roa katoa o te trachea i ia taahiraa, kia tino taupatupatu i waenga i ia whakaahua kia taea ai te "tuitui" whakaahua. Ko ngā whakaahua mai i ia trachea i Kātahi ka huihuia hei whakaahua kotahi mā te whakamahi i te Image Composite Editor v2.0.3 (Microsoft Research) mā te whakamahi i tētahi pūnaha nekehanga papatahi. I tauahia ngā rohe whakaaturanga LacZ i roto i ngā whakaahua whakakotahi o te trachea mai i ia kararehe mā te whakamahi i tētahi tuhinga MATLAB aunoa (R2020a, MathWorks) e ai ki te whakaahuatanga o mua, mā te whakamahi i ngā tautuhinga o 0.35 < Hue < 0.58, Saturation > 0.15, me te Value < 0.7. Mā te whai i ngā āhua o te kiko, i hangaia ā-ringa he kopare i roto i te GIMP v2.10.24 mō ia whakaahua whakakotahi hei tautuhi i te rohe kiko me te ārai i ngā kitenga teka mai i waho o te kiko tracheal. I whakarapopototia ngā rohe kua pania mai i ngā whakaahua whakakotahi katoa mai i ia kararehe hei whakaputa i te rohe kua pania katoa mō taua kararehe. Kātahi ka wehea te rohe kua pania ki te rohe kopare katoa hei whakaputa i te rohe kua whakatauritea.
I whakatōkia ia trachea ki roto i te paraffin, ā, i tapahia ngā wāhanga 5 μm. I pania ngā wāhanga ki te whero tere koretake mō te 5 meneti, ā, i tangohia ngā whakaahua mā te whakamahi i te karuārai Nikon Eclipse E400, te kāmera DS-Fi3 me te pūmanawa hopu huānga NIS (putanga 5.20.00).
I whakahaerehia ngā tātaritanga tatauranga katoa i roto i te GraphPad Prism v9 (GraphPad Software, Inc.). I whakatakotoria te hiranga tatauranga ki te p ≤ 0.05. I manatokohia te āhua noa mā te whakamahi i te whakamātautau Shapiro-Wilk, ā, i aromatawaihia ngā rerekētanga o te tae LacZ mā te whakamahi i te whakamātautau-t kore-tauira.
I tirotirohia ngā MP e ono i whakaahuatia i roto i te Ripanga 1 mā te whakamahi i te PCXI, ā, ko te tirohanga kei roto i te Ripanga 2. Kāore i kitea ngā MP polystyrene e rua (MP1 me MP2; 18 μm me 0.25 μm, ia) i raro i te PCXI, engari ko ngā tauira e toe ana i taea te tautuhi (kei te Whakaahua 5 ngā tauira). He iti te kitea o te MP3 me te MP4 (10-15% Fe3O4; 0.25 μm me 0.9 μm, ia). Ahakoa kei roto ētahi o ngā matūriki iti rawa i whakamatautauria, ko te MP5 (98% Fe3O4; 0.25 μm) te mea tino kitea. He uaua te kite i te hua CombiMag MP6. I ngā wā katoa, i tino whakarei ake tō mātou kaha ki te kite i te MP mā te whakawhiti i te aukume ki mua, ki muri hoki e whakarara ana ki te capillary. I te neke atu o ngā aukume i te capillary, ka toro atu ngā matūriki ki roto i ngā aho roa, engari i te tata haere o ngā aukume, ā, i te piki haere o te kaha o te mara aukume, ka poto haere ngā aho matūriki i te hekenga o ngā matūriki ki te mata o runga o te capillary (tirohia te Ataata Tāpiri S1: MP4), ka piki haere te te mātotoru o te matūriki o te mata. I tētahi atu taha, ina tangohia te aukume mai i te capillary, ka heke te kaha o te mara, ā, ka whakarite anō ngā MP ki ngā aho roa e toro atu ana mai i te mata o runga o te capillary (tirohia te Ataata Tāpiri S2:MP4). I muri i te mutu o te nekehanga o te aukume, ka haere tonu ngā matūriki ki te neke mō te wā poto i muri i te taenga ki te tūranga taurite. I te nekehanga o te MP ki te taha, ki te taha atu hoki i te mata o runga o te capillary, ka tōia e ngā matūriki aukume ngā paru mā roto i te wai.
He rerekē te kitea o te MP i raro i te PCXI i waenga i ngā tauira.(a) MP3, (b) MP4, (c) MP5 me (d) MP6. Ko ngā whakaahua katoa e whakaaturia ana i konei i tangohia mā te whakamahi i tētahi aukume kei te 10 mm pea i runga ake i te capillary. Ko ngā porowhita nui e kitea ana he mirumiru hau kua mau i roto i ngā capillary, e whakaatu mārama ana i ngā āhuatanga taha pango me te mā o te whakaahua rerekētanga ā-wāhanga. Kei roto i te pouaka whero te whakanui whakarei-rerekētanga. Kia mahara ko ngā diameter o ngā hoahoa aukume i roto i ngā pikitia katoa kāore i te tauinehia, ā, he tata ki te 100 ngā wā he nui ake i te mea e whakaaturia ana.
I te nekehanga o te aukume ki te taha maui me te taha matau i te tihi o te capillary, ka huri te koki o te aho MP kia rite ki te aukume (tirohia te Pikitia 6), ā, ka tautuhia ngā rārangi aukume. Mō te MP3-5, i muri i te taenga o te aho ki te koki paepae, ka tōia ngā matūriki i te mata o runga o te capillary. He maha ngā wā ka hua ake tēnei i te whakarōpūtanga o ngā MP ki ngā rōpū nui ake e tata ana ki te wāhi e kaha rawa ana te aukume (tirohia te Ataata Tāpiri S3:MP5). Ka kitea anō hoki tēnei ina whakaahuahia tata ki te pito capillary, ka huihui ngā MP me te arotahi ki te atanga wai-hau. Ko ngā matūriki i roto i te MP6, he uaua ake te kite i te MP3-5, kāore i tōia i te nekenga o te aukume i te capillary, engari ka wehea ngā aho MP, ka waiho ngā matūriki ki te tirohanga (tirohia te Ataata Tāpiri S4:MP6). I ētahi wā, i te wā i whakaitihia ai te aukume i whakamahia mā te neke i te aukume ki tawhiti atu i te wāhi whakaahua, ka heke mārire ngā MP e toe ana ki te mata o raro o te ngongo mā te kaha ā-papa i a rātou e noho ana i roto i te aho (tirohia te Ataata Tāpiri S5:MP3).
Ka huri te koki o te aho MP i te nekehanga o te aukume ki te taha matau i runga ake i te capillary.(a) MP3, (b) MP4, (c) MP5 me (d) MP6. Kei roto i te pouaka whero te whakanui whakarei-whakarerekētanga. Kia mahara he whai kōrero ngā ataata tāpiri nā te mea e whakaatu ana i te hanganga matūriki nui me ngā mōhiohio hihiri e kore e taea te tiro i roto i ēnei whakaahua tūmau.
I whakaatuhia e ā mātou whakamātautau mā te neke haere i te aukume mārire ki mua, ki muri hoki i te trachea ka taea te whakaatu i te MP i roto i te horopaki o te nekehanga uaua i roto i te tinana ora. Kāore i mahia te whakamātautau i roto i te tinana ora nā te mea kāore i kitea ngā pīrori polystyrene (MP1 me te MP2) i roto i te capillary. I whakamatautauria ia o ngā MP e whā e toe ana i roto i te tinana ora me te tuaka roa o te aukume i whirihorahia i runga ake i te trachea i te koki o te 30° ki te poutū (tirohia ngā Pikitia 2b me te 3a), nā te mea i hua ake he mekameka MP roa ake, ā, he pai ake i te whirihoranga aukume i mutu. Kāore i kitea te MP3, MP4 me te MP6 i roto i te trachea o tētahi kararehe ora. I te wā i whakaahuahia ai ngā ara manawa o te kiore i muri i te patunga tangata o ngā kararehe, i noho tonu ngā matūriki i te kore e kitea ahakoa i tāpirihia he rōrahi tāpiri mā te whakamahi i te papu weronga. Ko te MP5 te mea nui rawa atu o te waikura rino, ā, ko ia anake te matūriki e kitea ana, nō reira i whakamahia ai hei aromatawai me te whakaahua i te whanonga i roto i te tinana ora o te MP.
Nā te whakanoho i te aukume ki runga i te trachea i te wā e tukuna ana te MP, he maha ngā MP i kukū ki te tirohanga whānui, engari kāore i te katoa. Ko ngā matūriki e tomo ana ki te trachea ka kitea pai i roto i ngā kararehe i patua mā te tangata. Pikitia 7 me te Ataata Tāpiri S6: E whakaatu ana te MP5 i te hopu tere me te whakaritenga aukume o ngā matūriki i runga i te mata o te trachea ventral, e tohu ana ka taea te tuku i ngā MP ki ngā rohe e hiahiatia ana o te trachea. I te rapunga i tawhiti atu i te trachea i muri i te tukunga MP, i kitea ētahi MP e tata ana ki te carina, e kī ana kāore i te rawaka te kaha o te papa aukume ki te kohikohi me te pupuri i ngā MP katoa, nā te mea i tukuna mā te rohe o te kaha o te papa aukume mōrahi i te wā o te tukanga wai. Heoi anō, he teitei ake ngā kukū MP postpartum huri noa i te rohe whakaahua, e kī ana he maha ngā MP i noho ki ngā rohe manawa i te wāhi i teitei rawa ai te kaha o te papa aukume.
Ngā whakaahua mai i (a) i mua atu me (b) i muri i te tukunga o te MP5 ki roto i te trachea o tētahi kiore kua whakamatea tata nei me te aukume kei runga ake i te wāhi whakaahua. Kei waenganui i ngā mowhiti kiriuhi e rua te wāhi whakaahua. I mua i te tukunga o te MP, he wai kei roto i te ara manawa. Kei roto i te pouaka whero te whakanui whakarei-whakarerekētanga. Nō te ataata e whakaaturia ana i te Ataata Tāpiri S6:MP5 ēnei whakaahua.
Nā te whakawhiti i te aukume i te trachea in vivo i huri ai te koki o te mekameka MP i roto i te mata o te ara manawa, he rite ki te mea e kitea ana i roto i ngā capillaries (tirohia te Pikitia 8 me te Ataata Tāpiri S7:MP5). Heoi, i roto i tā mātou rangahau, kāore i taea te tōia ngā MP i te mata o te ara manawa ora pērā i tā rātou i tōia ai me ngā capillaries. I ētahi wā, ka roa ake te mekameka MP i te neke haere o te aukume ki maui, ki matau hoki. He mea whakamere, i kitea anō e mātou te āhua nei ka huri te aho matūriki i te hohonutanga o te paparanga wai mata ina nekehia te aukume ki te taha roa i te trachea, ā, ka whānui ina nekehia te aukume ki runga ake, ā, ka hurihia te aho matūriki ki te tūranga poutū (tirohia te Ataata Tāpiri S7). : MP5 i te 0:09, matau raro). I rerekē te āhua o te nekehanga i te wa i nekehia ai te aukume puta noa i te tihi o te trachea ki te taha (arā, ki te taha maui, ki te taha matau rānei o te kararehe, kaua ki te taha o te trachea). I kitea tonutia ngā matūriki i a rātou e neke ana, engari i te wa i tangohia ai te aukume mai i te trachea, ka kitea ngā pito o ngā aho matūriki (tirohia te Ataata Tāpiri S8:MP5, mai i te 0:08). He rite tonu tēnei ki te whanonga MP i kitea e mātou i raro i te papa aukume i whakamahia i roto i te capillary karāhe.
He tauira whakaahua e whakaatu ana i te MP5 i roto i te trachea o tētahi kiore ora kua whakamoea. (a) Ka whakamahia te aukume hei tiki whakaahua i runga ake nei, i te taha maui hoki o te trachea, kātahi (b) i muri i te nekehia o te aukume ki te taha matau. Kei roto i te pouaka whero te whakanui whakarei-whakarerekētanga. Nō te ataata e whakaaturia ana i te Ataata Tāpiri S7:MP5 ēnei whakaahua.
I te whirihoranga o ngā pou e rua ki te aronga raki-tonga i runga ake, i raro hoki i te trachea (arā, te kukume; Pikitia 3b), i puta ake ngā aho MP he roa ake, ā, kei te taha o te trachea, kaua ki te mata o te tracheal tuara (tirohia te Ataata Tāpiri S9:MP5). Heoi, kāore i kitea he nui ngā kukū o ngā matūriki i te wāhi kotahi (arā, te mata o te trachea tuara) i muri i te tukunga wai i te wā i whakamahia ai he taputapu aukume-rua, e puta ana i te nuinga o te wā ina whakamahia he taputapu aukume-kotahi. Kātahi ka whirihorahia tētahi aukume hei pana i ngā pou kua hurihia (Pikitia 3c), kāore te maha o ngā matūriki e kitea ana i te tirohanga i te āhua nei ka piki ake i muri i te tukunga. He uaua te whakatakotoranga o ngā whirihoranga aukume-rua e rua nā te kaha teitei o te papa aukume e kukume ana, e pana ana rānei i ngā aukume. Kātahi ka hurihia te whakatakotoranga ki tētahi aukume kotahi e whakarara ana ki te ara manawa engari e haere ana i roto i te ara manawa i te 90 nekehanga kia whiti ai ngā rārangi mara i te pakitara tracheal i te taha (Pikitia 3d), he whakatakotoranga i hangaia hei whakatau mēnā ka taea te kite i te kohikohinga matūriki i te pakitara taha. Heoi, i roto i i tēnei whirihoranga, kāore i kitea he nekehanga o te kohikohinga MP, te nekehanga aukume rānei. I runga i ēnei hua katoa, i whiriwhiria he whirihoranga aronga 30-tohu, kotahi-aukume (Pikitia 3a) mō ngā rangahau kawe ira in vivo.
I te wā i whakaahuahia ai te kararehe i muri tonu i te patunga tangata, nā te korenga o te nekehanga kiko whakararuraru i kitea ai ngā rārangi matūriki pai ake, poto ake i roto i te mara mārama i waenga i ngā chondral, "wiriwiri" i te taha o te nekehanga whakawhiti o te aukume. Ahakoa rā, kāore tonu e taea te kite mārama i te aroaro me te nekehanga o ngā matūriki MP6.
Ko te taitore LV-LacZ he 1.8 × 108 TU/ml, ā, i muri i te whakaranu 1:1 ki te CombiMag MP (MP6), i whiwhi ngā kararehe i te horopeta tracheal 50 μl o te 9 × 107 TU/ml LV vehicle (arā, 4.5 × 106 TU/kiore). I roto i ēnei rangahau, hei utu mō te whakamaoritanga i te aukume i te wā e whānau ana, i whakatūria e mātou te aukume ki te tūranga kotahi hei whakatau mēnā ka taea te whakapai ake i te whakawhiti LV (a) ki te whakataurite ki te tuku vector i te korenga o te papa aukume, me te (b) ka taea te arotahi Ka whakawhitihia ngā pūtau ara manawa ki ngā rohe ūnga aukume o te ara manawa o runga.
Ko te aroaro o ngā aukume me te whakamahinga o CombiMag me ngā kawe LV i te taha, kāore i te kitea he pānga kino ki te hauora o ngā kararehe, pērā i tā mātou tikanga tuku kawe LV paerewa. Ko ngā whakaahua o mua o te rohe tracheal i pangia e te raruraru miihini (Pikitia Tāpiri 1) i tohu he nui ake ngā taumata o te whakawhiti i roto i te rōpū kararehe i rongoatia ki te LV-MP i te wā i kitea ai te aukume (Pikitia 9a). He iti noa iho te tae kikorangi LacZ i kitea i roto i te rōpū whakahaere (Pikitia 9b). Ko te ine i ngā wāhi kua pania ki te X-Gal kua whakatauritea i whakaatu ko te whakahaerenga o te LV-MP i te aroaro o te papa aukume i hua ake he whakapainga tata ki te 6-ngā wā (Pikitia 9c).
Tauira whakaahua hiato e whakaatu ana i te whakawhiti ira ki te trachea e te LV-MP (a) i te aroaro o te papa aukume me te (b) i te ngaro o te aukume. (c) He whakapainga nui ā-tatauranga i roto i te rohe whakawhiti LacZ kua whakatauritea i roto i te trachea i te whakamahinga o te aukume (*p = 0.029, whakamātautau-t, n = 3 mō ia rōpū, toharite ± SEM).
I whakaatuhia e ngā wāhanga whero tere kūpapa (hei tauira i whakaaturia i te Pikitia Tāpiri 2) ngā pūtau kua pania ki te LacZ kei roto i te tauira me te wāhi rite tonu ki te pūrongo o mua.
Ko te wero matua mō te rongoā ira ara manawa ko te kimi tika i ngā matūriki kawe ki ngā rohe e hiahiatia ana, me te whakatutuki i ngā taumata teitei o te pai o te whakawhiti i roto i te pukahukahu neke i te aroaro o te rere o te hau me te whakakorenga hūpē kaha. Mō ngā kawe LV i hangaia hei rongoā i te mate ara manawa CF, ko te whakanui ake i te wā noho o ngā matūriki kawe i roto i ngā ara manawa he whāinga uaua ki te kitea. E ai ki a Castellani et al., he painga te whakamahinga o ngā papa aukume hei whakapai ake i te whakawhiti i ngā tikanga tuku ira kē atu pērā i te electroporation, nā te mea ka taea te whakakotahi i te māmā, te whai huatanga utu, te kimi i te rohe tuku, te pai ake o te mahi, me ngā wā whakatipu poto ake, me te iti ake pea o te horopeta kawe10. Heoi, kāore anō kia whakaahuahia te whakatakotoranga me te whanonga o ngā matūriki aukume i roto i ngā ara manawa i raro i te awe o ngā kaha aukume o waho, kāore anō hoki kia whakaaturia te whaihua o tēnei tikanga i roto i te tinana hei whakarei ake i ngā taumata whakaaturanga ira i roto i ngā ara manawa ora tonu.
I whakaatuhia e ā mātou whakamātautau PCXI synchrotron in vitro ko ngā matūriki katoa i whakamatautauria e mātou, haunga te polystyrene MP, i kitea i roto i te whakatakotoranga whakaahua i whakamahia e mātou. I te aroaro o te papa aukume, ka hangaia e ngā MP he aho e pā ana te roa ki te momo matūriki me te kaha o te papa aukume (arā, te tata me te nekehanga o te aukume). E ai ki te Pikitia 10, ko ngā aho e kitea ana e mātou ka hangaia nā te mea kua aukumetia ia matūriki takitahi, ā, ka puta ake tana ake papa aukume ā-rohe. Mā ēnei papa motuhake ka huihui, ka hono atu hoki ētahi atu matūriki rite, me ngā nekehanga rite ki te aho rōpū nā ngā kaha ā-rohe mai i ngā kaha kukume me ngā kaha whakahē ā-rohe o ētahi atu matūriki.
He hoahoa e whakaatu ana i ngā tereina matūriki (a,b) i hangaia i roto i ngā uaua toto kī tonu i te wai me te (c,d) trachea kī tonu i te hau. Kia mahara kāore ngā uaua toto me te trachea i tuhia ki te tauine. Kei roto hoki i te papa (a) he whakaahuatanga o te MP, kei roto ko ngā matūriki Fe3O4 kua whakaritea i roto i ngā aho.
I te nekehanga o te aukume ki runga ake i te capillary, ka eke te koki o te aho matūriki ki te paepae matua mō te MP3-5 kei roto ko te Fe3O4, ā, i muri iho ka mutu te noho o te aho matūriki ki te tūranga taketake, engari ka neke haere i te mata ki tētahi tūranga hou. aukume. Tērā pea ka puta tēnei pānga nā te mea he maeneene te mata o te capillary karāhe kia taea ai tēnei nekehanga. He mea whakamīharo, kāore te MP6 (CombiMag) i whanonga pēnei, nā te mea pea he iti ake ngā matūriki, he rerekē ngā paninga, he rerekē rānei ngā utu mata, he rerekē rānei te wai kawe i pā ki tō rātou kaha ki te neke. He ngoikore ake hoki te rerekētanga o te ahua o ngā matūriki CombiMag, e kī ana he rite tonu ngā matotoru o te wai me ngā matūriki, nō reira kāore e ngāwari te neke tetahi ki tetahi. Ka taea hoki e ngā matūriki te piri mena ka tere rawa te neke o te aukume, e tohu ana kāore e taea e te kaha o te papa aukume te wikitoria i te waku i waenga i ngā matūriki i roto i te wai, e kī ana pea ehara i te mea miharo ko te kaha o te papa aukume me te tawhiti i waenga i te aukume me te rohe ūnga he tino mea nui. Ina tangohia ngātahi, e kī ana hoki ēnei hua, ahakoa ka taea e ngā aukume te hopu i te maha o ngā MP e rere ana i roto i te rohe ūnga, kāore pea e taea te whakawhirinaki ki ngā aukume. hei neke i ngā matūriki CombiMag i te mata o te trachea. Nō reira, ko tā mātou whakatau me whakamahi ngā rangahau LV-MP in vivo i ngā papa aukume tūmau hei arotahi ā-tinana ki ngā rohe motuhake o te rākau ara manawa.
Ina tukuna ngā matūriki ki roto i te tinana, he uaua ki te tautuhi i roto i te horopaki o te kiko tinana e nekeneke ana, engari i whakarei ake te kaha ki te kite i a rātou mā te whakawhiti i te aukume whakapae ki runga ake i te trachea hei "whakakorikori" i ngā aho MP. Ahakoa e taea ana te whakaahua ora, he māmā ake te kite i te nekehanga o te matūriki ina oti te patu i te kararehe. Ko te nuinga o te wā ko te kukū MP te mea teitei rawa atu i tēnei wāhi i te wā i whakanohoia ai te aukume ki runga ake i te rohe whakaahua, ahakoa i kitea ētahi matūriki i te taha o te trachea. He rerekē ki ngā rangahau in vitro, kāore e taea te tōia ngā matūriki i te taha o te trachea mā te whakawhiti i te aukume. He rite tēnei kitenga ki te āhua o te tukatuka a te hūpē e kapi ana i te mata o te trachea i ngā matūriki e manawahia ana, ka mau ki roto i te hūpē, ā, muri iho ka horoia e te tikanga whakakorenga mucociliary.
I whakapae mātou mā te whakamahi i ngā aukume hei kukume i runga ake, i raro hoki i te trachea (Pikitia 3b) ka hua ake he papa aukume ōrite ake, kaua he papa aukume e tino kukū ana i te kotahi wāhi, ka arahi pea ki te tohatoha ōrite ake o ngā matūriki. Heoi, kāore i kitea e tā mātou rangahau tuatahi he taunakitanga mārama hei tautoko i tēnei whakapae. Waihoki, ko te whirihora i te takirua o ngā aukume hei pana (Pikitia 3c) kāore i hua ake he nui ake te whakatakotoranga matūriki i roto i te rohe whakaahua. E whakaatu ana ēnei kitenga e rua kāore te tatūnga aukume rua e whakapai ake i te mana whakahaere ā-rohe o te arotahi MP, ā, he uaua ki te whirihora i ngā kaha aukume kaha e puta mai ana, ka iti ake te whai hua o tēnei huarahi. Waihoki, ko te whakatakoto i te aukume ki runga ake, mā roto hoki i te trachea (Pikitia 3d) kāore hoki i whakanui ake i te maha o ngā matūriki e puritia ana i roto i te rohe whakaahua. Ko ētahi o ēnei whirihoranga rerekē kāore pea e angitu nā te mea ka hua ake he iti ake te kaha o te papa aukume i roto i te rohe whakatakotoranga. Nō reira, ko te whirihoranga aukume koki 30-tohu kotahi (Pikitia 3a) e kiia ana ko te tikanga māmā me te tino whai hua mō te whakamātautau in vivo.
I whakaatuhia e te rangahau LV-MP i te wā i whakakotahihia ai ngā kawe LV ki te CombiMag, ā, i tukuna i muri i te whakararuraru tinana i te aroaro o te papa aukume, i tino piki ake ngā taumata whakawhiti i roto i te trachea ki te whakataurite ki ngā mana whakahaere. I runga i ngā rangahau whakaahua synchrotron me ngā hua LacZ, i te āhua nei i taea e te papa aukume te tiaki i te LV i roto i te trachea me te whakaiti i te maha o ngā matūriki kawe i uru wawe ki roto i te pukahukahu. Mā ēnei whakapainga arotahi ka hua ake he whai huatanga teitei ake me te whakaiti i ngā taitara kua tukuna, te whakawhiti i waho o te ūnga, ngā pānga taha mumura me te ārai mate, me ngā utu kawe ira. He mea nui, e ai ki te kaihanga, ka taea te whakamahi i te CombiMag me ētahi atu tikanga whakawhiti ira, tae atu ki ētahi atu kawe huaketo (pēnei i te AAV) me ngā waikawa karihi.